The goal of this project is to use high throughput sequencing and computational analysis to gain a better understanding of the vls antigenic variation system, by which Lyme disease-causing bacteria evade the human immune system. The vls system is found in all Lyme disease bacteria and is required for evasion of adaptive immunity and survival of the organism beyond the first two weeks of infection. The vls locus consists of a single expression site encoding the surface lipoprotein VlsE and a contiguous array of silent DNA cassettes. Through a poorly understood gene conversion mechanism, random DNA segments from the silent cassettes replace the corresponding regions in the expression site, causing changes in the VlsE protein and evasion of the host antibody response. Immune system evasion leads to long-term infection and manifestations that may persist for months to years. Using DNA sequencing in combination with our analysis software, this project will provide the most comprehensive, statistically accurate analysis of the DNA recombination events in the vls antigenic variation system of Borrelia burgdorferi, a bacterium that causes Lyme disease. The resulting information will aid in understanding the processes involved in Lyme disease recombination events in mammalian hosts and evasion of host immune systems. Moreover, the approaches developed and demonstrated with the vls system can later be applied to other pathogenic systems which use similar tactics for evasion of the human and animal host immune systems. A thorough understanding of this variation system is important for the development strategies for interrupting the transmission cycle and preventing debilitating long-term manifestations of Lyme disease. The single aim of this research is to study a large number of DNA sequences (> 1.2 million) derived from animals with differing times of Lyme disease infection, and comparison to the original inoculates with which they were infected. The samples were previously collected from mice infected with Lyme disease causing clones for 7, 14, 28, and 90 days; samples from in vitro cultures and infected tissue explants will be included for comparison. PacBio DNA sequencing data will be aligned and analyzed using a novel sequence analysis algorithm (Varaligner) to provide an in-depth view of vlsE variant population present within each sample. Results obtained in different time points and tissues will be compared to assess the recombination patterns and kinetics, which may reveal the critical sequences involved in this gene conversion process. The software developed will be provided to the scientific community for similar in-depth analyses of antigenic variation systems in other bacterial and protozoal pathogens.